Transcript Leukocyte Differentiation Antigens Cell Adhesion Molecules NK cells

Leukocyte Differentiation Antigens
Cell Adhesion Molecules
NK cells
Linrong Lu (鲁林荣)
Institute of Immunology
Zhejiang University School of Medicine
Medical Research Building A801
Tel: 88981175
E-mail：[email protected]
Contents
• Concept of leukocyte differentiation antigens
（白细胞分化抗原）
• Concept of cluster of differentiation
（CD分子，分化群）
• Cell adhesion molecules
• NK cells
Leukocyte differentiation antigens
• Leukocyte differentiation antigens: Cell surface
molecules which may appear or disappear from the
cell membrane of leukocytes in the different
stages of differentiation and activation.
（第6版p58）
• Leukocyte differentiation antigens are also found
on cells other than leukocytes, such as
erythrocytes, endothelial cells, epithelial cells.
Classification of LDA
•
•
•
•
•
•
Immunoglobulin superfamily
Cytokine receptor family
C-Type lectin superfamily
Integrin family
TNF superfamily
TNF receptor superfamily
• Cluster of differentiation (CD): Cell surface molecules
can be recognized by particular monoclonal antibodies.
All of the monoclonal antibodies that react with a
particular membrane molecule are grouped together
as a cluster of differentiation (CD).
• Each new mAb that recognizes a cell surface molecule
is analyzed for whether it falls into a recognized CD
designation; if not, it is given a new CD designation
that reflects a new membrane molecule.
• CD1~CD363, up to 363 most recently (as of 2010) in
human
Human CD
Groups
T cell
CD（example）
CD2、CD3、CD4、CD5、CD8、CD28、CD152(CTLA4)、
CD154(CD40L)、CD272(BTLA)、CD278(ICOS)、CD294(CRTH2)
B cell
CD19、CD20、CD21、 CD40、CD79a(Ig)、CD79b (Ig)、
CD80 (B7-1)、CD86 (B7-2) 、CD267(TACI)、CD268(BAFFR)、
CD269(BCMA)、CD307(IRTA2)
Myeloid cell CD14、CD35(CR1)、CD64 (FcRI)、CD256(APRIL)、
CD257(BAFF)、CD312(EMR2)
Platelet CD36、CD41、CD42aCD42d、CD51、
CD61 、CD62P
NK cell CD16 (FcRIII) 、CD56(NCAM-1)、CD94、CD158(KIR)、
CD161(NKR-P1A)、CD314(NKG2D)、CD335(NKp46)、
CD336(NKp44)、CD337(NKp30)
Non-lineage CD30、CD32(FcRII)、CD45RA、CD45RO、CD46(MCP)、
CD55(DAF)、CD59、CD252(OX40L)、 CD279(PD1)、
CD281CD284(TLR1-TLR4)、CD289(TLR9)、 CD305(LAIR-1)、
CD306(LAIR-2)、CD319(CRACC)
Human CD
Groups
Adhesion molecules
CD（example）
CD11a-CD11c、CD15、CD15s(sLx)、CD18 、
CD29 、CD49aCD49f、CD54(ICAM-1)、
CD62E、CD62L 、CD324(E-cadherin)、
CD325(N-cadherin)、CD326(EpCAM)
Cytokine/
CD25(IL-2R)、CD95(Fas)、CD116-CDw137、CD178(FasL)、
Chemokine receptors
CD183(CXCR3)、CD184(CXCR4)、CD195(CCR5)、
CD261CD264(TRAIL-R1TRAIL-R4)
Endothelia
CD105(TGF-RIII)、CD106(VCAM-1)、CD140(PDGFR)、
CD144、CD299(DCSIGN-related)、
CD309(VEGFR2)、JAM1(CD321)、JAM2(CD322)
Carbohydrate structures CD15u、CD60aCD60c、CD75、CDw327CDw329
Dendritic cells
CD80,CD86,CD85
Stem cells
CD133、CD243
Erythrocytes CD233CD242
Stromal cells CD331-334 （FGFR1-FGFR4）
The CD system is commonly used
as cell markers
CD8
CD8
cytotoxic
Cell types and subtypes defined by CDs
Type of cell
CD markers
stem cells
CD34+,CD31-
all leukocyte groups
CD45+
Granulocyte
CD45+,CD15+
Monocyte
CD45+,CD14+
T lymphocyte
CD45+,CD3+
T helper cell
CD45+,CD3+,CD4+
Cytotoxic T cell
CD45+,CD3+,CD8+
B lymphocyte
CD45+,CD19+ or
CD45+,CD20+
Thrombocyte
CD45+,CD61+
Natural killer cell
CD16+,CD56+,CD3-
B cell
T cell
CD4+ T cell
Tregs
(CD4+CD25+)
Rules for assigning a CD number
• at least one Workshop—characterized antibody and
good molecular data.
• the antigen is expressed on the surface of cells
involved in immune reactions.
• the antibodies react with primary cells (not just
transfectant or recombinant protein).
• the antibody is available.
CD acts as Receptors or Ligands. Initiates a signal cascade, altering the behavior of
the cell, or has other functions, such as cell adhesion.
Functions of CDs
1.Mediate cell-cell interaction: Homing, activation, effecter functions
Functions of CDs
2. Receptors recognize exogenous or soluble substance: pathogens,
antibodies, cytokines
CDs related to T cell activation
TCR signaling
CD4 and CD8
•transmembrane glycoprotein
•co-receptor of T cell
T细胞
*CD4: first and second domains
bind to nonpolymorphic region of
MHC Ⅱmolecules
* CD8: IgV-like domain of  chain
binds to nonpolymorphic 3 region
of MHCⅠmolecules
•function
*for cell adhesion
*Involved in TCR-CD3 signal transduction
APC
Structure of CD4
Structure of CD8
CCR5
1.CD4 is a primary receptor used by HIV-1 to gain entry into host T cells.
2.HIV-1 attaches to CD4 with a protein in its viral envelope known as gp120. The binding
to CD4 creates a shift in the conformation of gp120 allowing HIV-1 to bind to other
surface receptors on the host cell, the chemokine receptors CCR5 .Following a structural
change in another viral protein (gp41), HIV inserts a into the host cell that allows the
outer membrane of the virus to fuse with the cell membrane.
3.HIV infection leads to a progressive reduction in the number of T cells possessing CD4
receptors. Therefore, medical professionals refer to the CD4 count to decide when to
begin treatment for HIV-infected patients. Normal blood values are 500-1200x106/L.
CD40 and CD40L (CD154)
BCR complex
co-receptor
CDs related to B cell activation
CD79 /CD79 
• Heterodimer
cytoplasmic domains
contain ITAM.
• Function:
1. transfers the signals
that lead to B cell
activation.
2. transports the Ig
BCR signaling
CD19/CD21/CD81
•
CD19/CD21/CD81 interactions with complement
associated with antigen play a role in antigeninduced B-cell activation.
•
CD21=CR2, C3dR, EBV R
CDs constituting FcR family
Different types of human Fc receptors
Role of FcRI (CD64) in the Ab-mediated
opsonization of microbes
Role of FcRIII (CD16) and FcRI in ADCC(antibodydependent cell-mediated cytotoxicity)
Cell adhesion molecules, CAM
• A group of proteins involved in adhesion
between cells or between cells and
extracellular matrix (ECM)
• CAMs and CD molecules are respectively
designated according to the different aspects of
these molecules.
• CAMs are grouped depending on their adhesion
functions.
• CD molecules contain the majority of CAMs that
have obtained CD designation. A minority of CAMs
have not been given CD designation.
Categories of CAM
1. IgSF (immunoglobulin superfamily)
2. Integrin family
3. Selectin family
4. Cadherin family
5. Others: CD44
Integrin
•
Integrin family is so named because the
molecules of this family primarily mediate the
cellular adherence to extracellular matrix,
enabling cells and extracellular matrix to form
the integration.
Structure:
Heterodimeric proteins consisting of two noncovalently bound polypeptides
( chain 1 out of 18, and  chain 1 out of 8).
Components of integrin family
1: VLA (very late activation antigen, 12 members),
ligand: VCAM-1(vascular cell adhsion molecule-1)
T cell activation (co-stimulation and adhesion)
2: LFA-1 (CD11a,lymphocyte function associated
antigen-1, 4 members);Mac-1(CD11b) ligand: ICAM1(intercellular adhesion molecule-1)
T cell activation, circulation, inflammation
3: gpIIb ligand:Fg (fibrinogen) Platelet aggregation
Distribution
Broad distribution on the surface of cells.
Selectin family
•
Selectin family includes L-selectin, P-selectin and
E-selectin, which play important roles in leukocyte
adhesion to endothelium, inflammation and
lymphocyte homing.
1. Structure
Transmembrane glycoproteins, with a number of
extracellular domains homologous to those seen in
complement control proteins (CCP). The extracellular
region also contains a domain related to the EGF
receptor and a distal C type lectin-like domain.
2. Members of selectin family
L-selectin: leukocytes (Cd62L)
P-selectin: platelets and megakaryocytes (Cd62P)
E-selectin: activated endothelia (Cd62E)
3. Ligand: carbohydrates, such as CD15s
Functions of CAM
1. Serving as co-receptors and providing co-stimulatory
signals in immune cells recognition and activation
CD4/CD8 -----MHC II/I (coreceptor)
CD28 -----B7 (costimulation)
2. Adhesion between leukocytes and vascular
endothelial cells in inflammation
3. Lymphocyte homing and recirculation
The sequence of events in the migration of blood leukocytes to sites of infection.
Selectins mediate weak tethering and rolling of blood neutrophils on the endothelium;
integrins mediate firm adhesion of neutrophils to the endothelium, and chemokines
activate the neutrophils and stimulate their migration through the endothelium to the
sites of infection.
Role of CAMs in migration of naïve and effector T cells. Naïve T cells home to
lymph node as a result of L-selectin binding to its ligand on HEVs(high-endothelial
venules), which are present only in lymph nodes. Activated T cells home to sites of
infection in peripheral tissues, and this migration is mediated by E-selectin, Pselectin and integrins.
Leucocytes adhesion to blood vessel
Application of CD, CAM and Their mAb
1. Elucidation of pathogenesis
CD4 and AIDS, gp120 binds to CD4
2. Application in diagnosis
leukemia, AIDS (ratio of CD4+ to CD8+ T cells)
3. Prophylactic and Therapeutic application
Graft rejection: Anti-CD3, Anti-CD25
non-Hodgkin’s lymphoma: Anti-CD20
Natural Killer cells
NK cells - What are they?
Human CD3-,CD56+
Mouse CD3-,NKp46+ (in B6 mice NK1.1+)
• 3rd lineage of lymphocytes
• Function in innate immunity to protect against
viruses, bacteria, & tumors
• Produce cytokines & kill abnormal cells
NK Cells - Where do they come from?
• Progenitor in bone marrow
• Thymus not required
– nude mice have normal NK cells
• Do not rearrange ,, or d-TcR or Ig
– normal NK cells in scid and Rag-/- mice
NK Cells - Where do they live?
• ~5-20% peripheral blood lymphocytes
• ~5% lymphocytes in spleen
• Abundant in liver
• Low frequency in thymus, bone marrow,
uninfected lymph nodes and lymphatics
• >90% of lymphocytes in decidual tissue
NK Cells - What do they do?
• Cell mediated-cytotoxicity
• Antibody-dependent cellular cytotoxicity
(ADCC)
• Early -interferon production
• Secretion of TNF, LT, GM-CSF, IL-5, MCSF, IL-3, IL-10, IL-13, MIP-1, MIP-1,
RANTES, etc.
NK cells- How do they kill?
• Predominantly perforin / granzymes
– Kagi et al Nature 369:31, 1994
•
Secreted or membrane TNF
– Degliantoni et al J Exp Med 162:1512 1985
•
Fas ligand
– Arase et al J Exp Med 181:1235, 1995
•
TRAIL
– Zamai et al J Exp Med 188:2375, 1998
NK cells - Cytokine Activation
**Interferon-/
– augments cytolytic activity
**IL-15
– required for development, induces proliferation, increases
cytotoxicity
IL-12 & IL-18
– augments INF production
IL-2
– induces proliferation, increases
cytotoxicity…..physiological relevance?
NK cells - what are they good for?
Protection from viruses & tumors
What are NK cells are good for?
Humans lacking NK cells susceptible to Varicella zoster &
CMV
–
Biron et al. N Engl J Med 320:1731 1989
Mouse NK cells protect against CMV - requires perforin
and IFN
–
Bukowski et al. J Immunol 131:991 1983, J Exp Med 161:40 1985; Welsh et al. J Exp Med
173:1053, 1991; Scalzo et al. J Exp Med 171:1469, 1990; Orange et al. J Exp Med
182:1045, 1995
Mouse NK cells protect against mousepox - requires IFN
–
Jacoby et al. Arch. Virol. 108:49, 1989; Chaudhri et al. PNAS 101:9057, 2004; Fan et al.
PLoS Pathogen 8:e30, 2008
Mouse NK cells protect against Ebola virus - requires
perforin, not IFN
–
Warfield et al J Exp 200:169, 2004
NK Cells Reject Tumors Lacking MHC Class I
Class I + tumors
grow in vivo
Class I- tumors
are rejected
Class I- tumors
in NK-depleted
mice grow in vivo
Immune surveillance for
‘ Missing Self’
NK cells preferentially kill cells that have lost MHC
class I
Provides protection against cells escaping T cell
recognition （tumor and viral-infected cells tend to
reduce the expression of MHC I to avoid the attack
from T cells）
Predicts existence of inhibitory receptors for MHC
class I that spare normal cells from NK cell attack
Karre et al. Nature 319:675, 1986
RBC, neural cells
Normal healthy cells
Virus-infected cells, tumor
Stressed cells
Virus-infected cells, tumor
Human Chr
19q13
Killer cell Ig-like Receptors (KIR)
Activating & inhibitory
NK receptors
Killer Cell Ig-like Receptors (KIR)
Ig superfamily
7-12 functional genes on human chromosome 19q13
Extensive allelic polymorphism (no rearrangement)
Mono-allelic expression possible
Inhibitory KIR recognize polymorphic HLA-A, -B, and –C
Activating receptors have no intrinsic signaling capacity..associate
with DAP12 ITAM-adapter protein
Expressed by subsets of NK cells and memory T cells (usually CD8+ T
cells)
Human NK Receptors for HLA class I
Human Receptors Ligands
KIR3DL3
?
KIR2DL3
HLA-CS77N80
KIR2DL2
HLA-CS77N80
KIR2DL1
HLA-CN77K80
KIR2DL4
HLA-G?
KIR3DL1
HLA-Bw4
KIR3DS1
?
KIR2DL5A/B
?
KIR2DS3
?
KIR2DS5
?
KIR2DS1
HLA-Cwe akly
KIR2DS2
?
KIR2DS4
HLA-Cwe akly
KIR3DL2
HLA-A?
CD94/
NKG2A
HLA-E
C-lectin domain
Ig-domain
CD94/
NKG2C
HLA-E
LILRB1
HLA-A,B,C,E,F,G
ITIM
K/R
Uhrberg et al. 1997; Wilson et al. 2000; Vilches et al. 2000; Hsu et al. 2002
Human KIR Haplotypes Differ in
Gene Content and Organization
3DL1
3DL3
2DL3
2DS2
2DL2
2DL5B
2DS3
2DP1
2DL1
3DP1
2DL4
2DL5A
2DS4
2DS5
2DS1
3DL2
3DS1
A haplotype
3DL3
2DL3
2DL1
2DL4
3DL1
2DL4
3DS1
2DS4
3DL2
B haplotype
3DL3
2DS2
2DL2
2DL1
2DS5
2DL5A
2DS1
3DL2
Generated by gene conversion - exon swapping
Difficult to distinguish loci versus alleles
Group A haplotype
3DL3
2DL3
55
2DL1
7
# alleles:
2DL4
15
2DS4
3DL1
26
49
3DL2
11
3 x 106 possible combinations in the group A haplotypes
(Courtesy P. Parham)
38
Mice don’t have KIR genes
Mice have Ly49 genes that do the same job
The "NK Complex" on Mouse Chromosome 6
and Human Chromosome 12p12-p13
Mouse
NKR-P1A,B,C CD94 NKG2D,C,A
Ly49A-I
Human
NKR-P1A
CD94
NKG2D,F,E,C,A Ly49L
Humans don’t have functional Ly49 genes
Ly49 Receptors
C-type lectin-like superfamily
Polygenic & polymorphic
Extensive allelic polymorphism (no rearrangement)
Inhibitory Ly49 recognize polymorphic H-2D and H-2K
Activating receptors have no intrinsic signaling
capacity..associate with DAP12 ITAM-adapter protein
Expressed by subsets of NK cells and memory T cells (usually
CD8+ T cells)
Inhibitory and activating Ly49 receptors
Ligand = MHC class I
Ly49A,C,G,I...
Ly49D,H
DAP12
-
ITIM
Recruit phosphatases inhibit responses
--+
+
ITAM
Recruit Syk & Zap70
kinases - activate
NKG2D
– C-type lectin-like superfamily
– 1 gene, non-polymorphic, conserved mice - humans
– Homodimer expressed on all NK cells, d T cells, and CD8+ T cells
– R in transmembrane associates with D in DAP10 transmembrane
DAP10
– 10 kd homodimer
– Cytoplasmic YINM recruits Grb2 & p85 PI3-kinase
NKG2D ligands in mice and humans
Many genes
Many alleles
NKG2D ligands
• MHC class I-like
– don’t require peptide or 2-microglobulin
• Bind with nM affinity to NKG2D
• Low levels expressed on healthy tissues
• Induced on virus-infected cells and tumor cells
• Induced by DNA damage
• Elevated in autoimmune diseases
What is the biological role of the
NKG2D ligands?
“Danger signals” to alert the
immune system to infection or
genomic damage
NK cells - Influence on
Adaptive Immunity
Early INF production by NK cells may ‘skew’
CD4+ T cells towards Th1
INF production by NK cells may cause IgG
class switching in B cells
Cross-talk with dendritic cells